The main focus of this research is to study RNA localization in leech embryos and to understand its role in[unreadable] normal embryonic development. RNA localization provides a mechanism for cells to specify the correct[unreadable] location for protein synthesis. RNA localization has been identified in embryonic and adult cells, and in[unreadable] organisms such as yeast, and several invertebrate, and vertebrate species. RNA localization, regardless of[unreadable] the type of cell or species, has several features in common. It requires the interaction between RNA-binding[unreadable] proteins and specific mRNA sequences, mainly on its 3' untranslated region (UTR). The transport and[unreadable] anchorage of mRNA is dependent on the cytoskeleton. In many organisms studied, the 3' UTR contains[unreadable] specific sequences involved in anchorage, transcriptional stability, translational regulation, and localization.[unreadable] All four functions (stability, translational regulation, localization, and anchorage) are important, but it is not[unreadable] clear if they affect each other. The long-term objective of the proposed research is to understand how mRNA[unreadable] translational repression/derepression, and stability are used by cells, to regulate proper mRNA localization[unreadable] and anchorage. The specific aims of this pilot project are to functionally characterize localization and[unreadable] anchorage sites found in the Hro-Twist (leech transcript) 3' UTR. Four approaches will be used. First, GFPHro-[unreadable] Twist 3' UTR deletion constructs will be obtained to verify preliminary studies. Second, these clones will[unreadable] be transcribed in vitro and injected in the zygote, to test for proper mRNA localization and mRNA anchorage[unreadable] in vivo. Third, a LacZ/Hro-Twist-3' UTR reporter construct will be used, to test prospective sequences[unreadable] involved in stability regulation. Fourth, PCR mutagenesis of ACE2 and ARE2 elements will be performed, to[unreadable] find those which are critical for function. Based on preliminary studies, we hypothesize that ACE2 elements[unreadable] in the Hro-Twist coding region are required to localize this transcript to the CD cell, and that ARE2 elements[unreadable] found in the 3' UTR are required for anchorage to the teloplasm.